Trehalose Toxicity in Cuscuta reflexa

a,a-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta relexa Roxb. vine. The incorporation of radioactivity from [I'C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confied to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A3, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxygucose, which has been shown to be a potent inhibitor of ceD wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of ceDl wail polysaccharides, the chief component of which was presumed to be cellulose.

A rapid and sensitive method for the estimation of individual tRNA pools

A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: (i) charging of total tRNA with a mixture of radiolabeled aminoacids, (ii) deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, (iii) derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, (iv) baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and (v) quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content. PSG, posterior silk gland; PITC, phenylisothiocyanate; DMAA, N,N-dimethyl-N-allylamine; APH, algal protein hydrolysate; ptc-, phenylthiocarbamyl; HPLC, high performance liquid chromatography.

Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Characterization of a toxin from Parthenium hysterophorus and its mode of excretion in animals

Fractionation of methanolic extracts of air dried aerial parts ofParthenium resulted in the isolation of a toxic constituent which was identified as parthenin, the major sesquiterpene lactone from the weed. The LD50 (minimal lethal dose required to cause 50% mortality) for parthenin in rats was 42 mg/kg body weight. When [3H]-parthenin was given orally or by intravenous administration, radioactivity appeared in the milk of lactating laboratory and dairy animals. Tissue distribution of radioactivity revealed that maximum label was detectable in kidneys.

Enzymatic acylation of 1-alkyl-, 1-alkenyl-and 1-acyl glycero-3-phosphorylethanolamine in developing rat brain

Rat brain particulate fractions were shown to acylate [32P]1-alkyl-sn-glycero-3-phosphorylethanolamine (GPE). While the main product is 1-alkyl-2-acyl GPE, about 12 per cent of the radioactivity was also found in 1-alkenyl-2-acyl GPE. The acyl transferase activity was completely dependent on added ATP and CoA and it was localized mainly in the microsomal fraction. A comparative study of acyl transferase activities to 1-alkyl-, 1-alkenyl-, and 1-acyl GPE by crude mitochondrial fraction and microsomes of 10, 16 and 22-day-old rat brains showed a progressive increase in activity with development. In the 22-day-old rat brain the order of activity towards the three substrates is as follows: 1-acyl GPE ± 1-alkenyl GPE ± 1-alkyl GPE with a crude mitochondrial fraction and 1-acyl GPE ± 1-alkyl GPE ± 1-alkenyl GPE with microsomes.

Number and proportion of selenocucleosides in the transfer RNA of escherichia-coli

tRNA isolated from escherichia-coli grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

F-18 Labelling Synthesis, Radioanalysis and Evaluation of A Dopamine Transporter and A Hypoxia Tracer

Positron emission tomography (PET) is an imaging technique in which radioactive positron-emitting tracers are used to study biochemical and physiological functions in humans and in animal experiments. The use of PET imaging has increased rapidly in recent years, as have special requirements in the fields of neurology and oncology for the development of syntheses for new, more specific and selective radiotracers. Synthesis development and automation are necessary when high amounts of radioactivity are needed for multiple PET studies. In addition, preclinical studies using experimental animal models are necessary for evaluating the suitability of new PET tracers for humans. For purification and analysing the labelled end-product, an effective radioanalytical method combined with an optimal radioactivity detection technique is of great importance. In this study, a fluorine-18 labelling synthesis method for two tracers was developed and optimized, and the usefulness of these tracers for possible prospective human studies was evaluated. N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([18F]β-CFT-FP) is a candidate PET tracer for the dopamine transporter (DAT), and 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is a well-known hypoxia marker for hypoxic but viable cells in tumours. The methodological aim of this thesis was to evaluate the status of thin-layer chromatography (TLC) combined with proper radioactivity detection measurement systems as a radioanalytical method. Three different detection methods of radioactivity were compared: radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography. The fluorine-18 labelling synthesis for [18F]β-CFT-FP was developed and carbon-11 labelled [11C]β-CFT-FP was used to study the specificity of β-CFT-FP for the DAT sites in human post-mortem brain slices. These in vitro studies showed that β-CFT-FP binds to the caudate-putamen, an area rich of DAT. The synthesis of fluorine-18 labelled [18F]FMISO was optimized, and the tracer was prepared using an automated system with good and reproducible yields. In preclinical studies, the action of the radiation sensitizer estramustine phosphate on the radiation treatment and uptake of [18F]FMISO was evaluated, with results of great importance for later human studies. The methodological part of this thesis showed that radioTLC is the method of choice when combined with an appropriate radioactivity detection technique. Digital PSL autoradiography proved to be the most appropriate when compared to the radioactivity scanning and film autoradiography methods. The very high sensitivity, good resolution, and wide dynamic range of digital PSL autoradiography are its advantages in detection of β-emitting radiolabelled substances.

Number and proportion of selenocucleosides in the transfer RNA of Image Image

tRNA isolated from . grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

Separation of 35S-Labeled thionucleosides of Escherichia coli and Pseudomonas aeruginosa transfer RNAs on a phosphocellulose column

35S-Labeled thionucleosides prepared from Escherichia coli and Pseudomonas aeruginosa tRNAs were chromatographed separately on a phosphocellulose column with a linear salt gradient of 0.005–0.1 M ammonium formate (pH 3.9). The thionucleosides of E. coli tRNA were quantitatively separated into four peaks which were identified using authentic samples as 4-thiouridine (78 %), 2-methylthio-N6-isopentenyladenosine (8 %), 2-thiocytidine (2.5 %) and 5-methylaminomethyl-2-thiouridine (11.5 %). In the case of P. aeruginosa tRNA four radioactive thionucleoside peaks were also observed. One major difference was the almost complete absence of 2-methylthio-N6-isopentenyladenosine and the presence of a new peak of radioactivity in the nucleosides of P. aeruginosa. The relative proportions of the various thionucleosides were found to be different in E. coli and P. aeruginosa tRNAs.

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv.

Two methods were employed to measure the rate of ribonucleic acid (RNA) chain growth in vivo in Mycobacterium tuberculosis H37Rv cultures growing in Sauton medium at 37 degrees C, with a generation time of 10 h. In the first, the bacteria were allowed to assimilate [3H]uracil or [3H]guanine into their RNA for short time periods. The RNA was then extracted and hydrolyzed with alkali, and the radioactivity in the resulting nucleotides and nucleosides was measured. The data obtained by this method allowed the calculation of the individual nucleotide step times during the growth of RNA chains, from which the average rate of RNA chain elongation was estimated to be about 4 nucleotides per s. The second method employed the antibiotic rifampin, which specifically inhibits the initiation of RNA synthesis without interfering with the elongation and completion of nascent RNA chains. Usint this method, the transcription time of the 16S, 23S, and 5S ribosomal RNA genes was estimated to be 7.6 min, which corresponds to a ribosomal RNA chain growth rate of 10 nucleotides per s.

Auger-emitter radiochemotherapy in squamous cell cancers : in vitro and in vivo experiments with In-111-BLMC

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in combined modality therapy (surgery, radiotherapy, chemotherapy) the 5-year survival rate in stage III and IV disease remains at 40% - 60%. Short-range Auger-electron emitters, such as In-111 and In-114m, tagged with a drug, molecule, peptide, protein or nanoparticles brought in close proximity to nuclear DNA represent a fascinating alternative for treating cancer. In this thesis, we studied the usefulness of Indium-111-bleomycin complex (In-111-BLMC) in the diagnostics and potential therapy of HNSCC using in vitro HNSCC cell lines, in vivo nude mice, and in vivo HNSCC patients. In in vitro experiments with HNSCC cell lines, the sensitivity to external beam radiation, BLM, In-111-BLMC, and In-111-Cl3 was studied using the 96-well plate clonogenic assay. The influence of BLM and In-111-BLMC on the cell cycle was measured with flow cytometry. In in vivo nude mice xenograft studies, the activity ratios of In-111-BLMC were obtained in gamma camera images. The effect of In-111-BLMC in HNSCC xenografts was studied. In in vivo patient studies, we determined the tumor uptake of In-111-BLMC with gamma camera and the radioactivity from tumor samples using In-111-BLMC with specific activity of 75, 175, or 375 MBq/mg BLM. The S values, i.e. absorbed dose in a target organ per cumulated activity in a source organ, were simulated for In-111 and In-114m. In vitro studies showed the variation of sensitivity for external beam radiation, BLM, and In-111-BLMC between HNSCC cell lines. IC50 values for BLM were 1.6-, 1.8-, and 2.1-fold higher than In-111-BLMC (40 MBq/mg BLM) in three HNSCC cell lines. Specific In-111 activity of 40 MBq/mgBLM was more effective in killing cells than specific In-111 activity of 195MBq/mgBLM (p=0.0023). In-111-Cl3 alone had no killing effect. The percentage of cells in the G2/M phase increased after exposure to BLM and especially to In-111-BLMC in the three cell lines studied, indicating a G2/M block. The tumor-seeking behavior was shown in the in vivo imaging study of xenografted mice. BLM and In-111-BLMC were more effective than NaCl in reducing xenografted tumor size in HNSCC. The uptake ratios received from gamma images in the in vivo patient study varied from 1.2 to 2.8 in malignant tumors. However, the uptake of In-111-BLMC was unaffected by increasing the injected activity. A positive correlation existed between In-111-BLMC uptake, Ki-67/MIB activity, and number of mitoses. Regarding the S values, In-114m delivered a 4-fold absorbed radiation dose into the tumor compared with In-111, and thus, In-114m-BLMC might be more effective than In-111-BLMC at the DNA level. Auger-electron emitters, such as In-111 and In-114m, might have potential in the treatment of HNSCC. Further studies are needed to develop a radiopharmaceutical agent with appropriate physical properties of the radionuclide and a suitable carrier to bring it to the targeted tissue.

Anthranilic acid hydroxylase from Aspergillus niger

The in vivo conversion of radioactive tryptophan to anthranilic acid and 2,3-dihydroxybenzoic acid by submerged cultures of Claviceps paspali was shown by Groeger and his co-workers (1965). More recently, Subba Rao et al. (1967a) reported that washed mycelial felts of Aspergillus niger incorporate the radioactivity from DL-tryptophan-C14 (benzene ring-labeled) into anthranilic acid, 3-hydroxyanthranilic acid, 2,3-dihydroxybenzoic acid and catechol. However, the conversion of anthranilic acid to 2,3-dihydroxybenzoic acid by cell-free preparations has not been demonstrated. In the present paper we report the demonstration of a soluble anthranilic acid hydroxylase from Aspergillus niger which is different from the anthranilic acid hydroxylases reported so far from microbes and higher plants.

Accumulation of glycine in the fat body of the silkworm, bombyx mori L. [31]

INVESTIGATIONS of intestinal transport of amino-acids in the locust1,2 and silkworm3,4 have shown no evidence for active accumulation in a transport from the insect gut of amino-acids. When glycine-2-14C was administered in vivo to fifth instar larvae of the silkworm, 96 per cent of the radioactivity was incorporated into various tissues within 1 h whereas in vitro only 19 per cent of the activity was transported by the mid-gut of silkworm (unpublished work). These results suggested that continued absorption of glycine by the intestine could be aided by a facilitated diffusion mechanism in which amino-acids are rapidly removed from the site of absorption either by accumulation into other tissues or by degradation. Although the insect fat body has been assigned both accumulatory and dissimilatory roles5, the mechanism of accumulation of amino-acids has not been investigated. Our present experiments show that the silkworm fat body possesses an efficient mechanism for accumulating glycine and that both the accumulation and the release of glycine are metabolically controlled.

Biosynthesis of coenzyme Q in microorganisms

Incorporation of mevalonate-2-C14, acetate-1-C14, and formate-C14 into the lipids of microorganisms was studied. In the case of four bacteria tested—Agrobacterium tumefaciens, Azotobacter vinelandii, Escherichia coli, and a Pseudomonas species—the various homologues of coenzyme Q present were not labeled with any of the tracers used, although significant amounts of radioactivity were present in the lipids. Both acetate and mevalonate were incorporated into coenzyme Q and sterol of the moulds, Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Gibberella fujickuroi, and a yeast, Torulopsis utilis. Mevalonate was incorporated into the side chain but not the ring, whereas acetate was incorporated into both. It appears that the mevalonate pathway for the synthesis of coenzyme Q is operative only in those organisms which also contain other isoprene compounds such as sterol and carotene.